Superoxide dismutases (SODs) are produced in almost all aerobic organisms, obligate anaerobes, and aero-tolerant anaerobes as a result of biological oxidations. These enzymes are found in almost all parts of the cell (Fink and Scandalios, 2002; Talukdar and Talukdar, 2013). Plant SOD proteins are a type of antioxidant enzyme that has been linked to protecting plants from the harmful effects of ROS and thus have a significant impact on plant growth, development, and their responses to abiotic stress (Rasul et al., 2017; Wang et al., 2017; Zhang et al., 2021a). In the natural environment, plants are frequently subjected to different environmental stressors, which result in the overproduction of ROS, such as singlet oxygen, hydrogen peroxide (H₂O₂), peroxyl radicals, hydroxyl radicals, and superoxide anion radicals. The controlled synthesis of cellular H₂O₂ serves a vital physiological function, but excessive amounts can have carcinogenic effects and induce cell death, and are produced in response to environmental stimuli in order to protect plant cells from O₂ toxicity. Lowering its concentration can increase plant growth and quality, as well as antioxidant enzyme activity. This ROS causes degradation of cell membrane, macromolecules, and peroxidation, eventually leading to cell death and hence limiting crop production worldwide (Dehury et al., 2013). Plants have created a range of enzymatic as well as non-enzymatic defense systems to adapt to these ROS, including SOD system, glutathione reductase system, glutathione S-transferase system, ascorbate peroxidase (APX) system, glutathione peroxidase (GPX) system, peroxiredoxin (PrxR) system, dehydroascorbate reductase system, and catalase (CAT) system (Wang et al., 2017; Zhou et al., 2017; Lu et al., 2020). Metalloenzymes, commonly referred to as SOD, are the first line of defense for organisms against hazardous ROS. SODs catalyze the disproportionate conversion of O₂ into oxygen and H₂O₂. Consequently, almost all green living cells are protected from the oxidative stress caused by aerobic circumstances (Dehury et al., 2013; Geng et al., 2018).

The SODs are divided into three classes based on their metal cofactors: copper/zinc SOD (Cu/Zn SOD or CSD), manganese SOD (MnSODs or MSD), and iron SOD (FeSODs or FSD) (Dreyer and Schippers, 2018). SOD proteins, which are encoded by nuclear genes, are found in several regions of the cell, with CSDs being found in the cytosol, chloroplast, and mitochondria (Feng et al., 2015; Verma et al., 2019). MSDs are thought to be found in peroxisomes as well as mitochondria, while FSDs are believed to be found in peroxisomes, chloroplast, and mitochondria (Verma et al., 2019). MSD and FSD have a high degree of sequence similarity, and most fungi and animals contain these two SODs, whereas there is no significant similarity in the case of CSD. Due to zinc and copper-binding as well as oxidation of intermolecular disulfide, CSD is generally highly stable. Copper is involved in superoxide disproportionation as a catalyst, while zinc and disulfide are involved in protein folding. Bacteria and plants exhibit all three types of SODs (Kliebenstein et al., 1998; Talukdar and Talukdar, 2013). In addition, Streptomyces were the first source for the identification, characterization, and cloning of a new form of SOD called nickel SOD or NiSOD. In contrast, plants have not been shown to contain these NiSODs (Hu et al., 2019).

The superoxide dismutase gene family has been found in a variety of plants, including Arabidopsis (Kliebenstein et al., 1998), Zostera marina (Zang et al., 2020), Oryza sativa (Dehury et al., 2013), Gossypium hirustum (Wang et al., 2017), grape (Vitis vinifera L.) (Guo et al., 2020), Medicago truncatula (Song et al., 2018), watermelon and melon (Zhang et al., 2021a), maize (Zea mays L.) (Sytykiewicz, 2014), and Rosaceae species (Li et al., 2021). Studies on different plant SOD genes have shown that these can act as the first line of defense against abiotic stimuli including heat, cold, drought, and salinity (Zhang et al., 2021a,b). The expression pattern of individual SOD gene family members is different in different species. This expression of individual members also varies significantly according to environmental stresses. In A. thaliana, the level of transcription of chloroplastic FSD1 and CSD2 were reduced by ozone fumigation, while chloroplastic FSD2 mRNAs remained relatively constant. However, FSD2 mRNA increased considerably in response to UV-B, but FSD1 and CSD2 remained stable. Furthermore, CSD1 may be involved in response to both UV-B and ozone illumination (Kliebenstein et al., 1998). In legumes, the overexpression of antioxidant enzymes may provide additional expression throughout the N₂ fixation, particularly under stress and senescence. Overexpression of MnSOD from Nicotiana plumbaginifolia protected alfalfa plants from water deprivation. Furthermore, overexpression of MnSOD in plants improved FeSOD activity (Kuzniak, 2002; Rubio et al., 2002).

Under drought stress, transgenic potato (carrying CSD cloned from Potentilla atrosanguinea) demonstrated higher stomatal conductance and photosynthetic rates as compared to the other wild-type plants. Similarly, more transcripts were produced by the white clover FSD, MSD, and CSD genes in drought stress. While banana SOD genes were downregulated, SOD gene expression from tomatoes was altered (Pal et al., 2013; Feng et al., 2015, 2016; Zhang et al., 2015). The expression of three types of SOD genes showed significant upregulation in G. hirsutum during drought stress; however, only the expression of CSD genes was increased under cold stress and the soluble sugar and photosynthetic rate were upregulated. Under cold stress, the concentration of ROS was higher with decreased SOD activity in tea plants, and damage to leaves was more severe in cold-sensitive cultivars than in cold-resistant cultivars (Zhang et al., 2014; Zhou et al., 2019). In wheat, members of this family showed different expressions in various tissues. Under heat, drought, and salt stress, wheat CSDs and MSDs were highly upregulated (Zafra et al., 2018). In Z. marina, all the five SOD genes were expressed in various tissues (leaf, root, male flower, female flower—early and late tissues). Consequently, boosting SOD activity is one of the most effective strategies for plants to withstand a variety of abiotic stresses (Talukdar and Talukdar, 2013).

Daucus carota (carrot) subsp. Carota L (2n = 2x = 18) is one of the most important economical crops globally and the most important member of the Apiaceae family. D. carota is one of the most essential vegetables in the world, and it comes in a variety of colors, including yellow, red, orange, white, and purple-pink, with its root rich in alpha and beta-carotene and a good source of vitamin K and B6 (Zhou et al., 2019). The most well-known feature of D. carota is that it was the first species to successfully demonstrate the integrity of living cells. Furthermore, experiments provided clues that altering DNA levels per cell leads to differential genome organization. Similarly, the significance of plastid transformation has been proven in several metabolic, biochemical, electron-microscopic, and histochemical studies. This reproducible system was crucial in elucidating the connection between genome organization and growth regulators and is still used for recent molecular biology research. Similarly, cell programming in D. carota is particularly simple at the tissue and cellular levels. These features make D. carota an ideal system for fundamental studies.

Superoxide dismutase genes help enhance food production by resisting a variety of environmental challenges (salt, drought, alkali, light/dark, and cold stress). Furthermore, the evolution and diversity in this gene family may lead to functional diversity, which may help to further understand the carrot stress-responsive genes. Therefore, to address whether SOD genes are conserved in D. carota, we identified these genes in carrots. In this study, we determined the functional diversity and expression profiles of DcSOD genes, which provides useful information for stress-resistant carrot breeding. This comprehensive investigation of the DcSOD genes will also serve as a foundation for future research into the molecular functions of DcSODs.

Plants have a number of effective enzymatic antioxidant defense mechanisms that prevent plant cells from oxidative damage by removing ROS. SOD is one of these antioxidant enzymes that are involved in a variety of plant functions, including growth and providing resistance to environmental stresses by acting as the first line of defense against the harmful effects of high levels of ROS (Feng et al., 2016; Zang et al., 2020; Zhang et al., 2021a). In this study, a genome-wide systematic analysis of the SOD gene family, including sequence phylogeny, conserved motifs, gene structure, chromosomal localization, expression profiling, and structure prediction was carried out in D. carota. The SOD gene family is widespread in a variety of plant species such as Pyrus bretschneideri, Rosaceae species (Guo et al., 2020), Olive (Olea europaea L.) (Zafra et al., 2018), Larix kaempferi (Han et al., 2019), barley (Hordeum vulgare L., Hv) (Zhang et al., 2021b), Populus (Molina-Rueda et al., 2013), Setaria italica (Wang et al., 2018), and S. miltiorrhiza (Han et al., 2020). In S. Italica and H. vulgare, SODs were overexpressed under salt stress and drought stress. In melon (C. melo) and watermelon (C. lanatus), overexpression of SODs under low temperature and salt stress confers resistance to various abiotic stresses (Geng et al., 2018). In cassava (Manihot esculenta Crantz), SOD with other enzymes was found to provide resistance against T. cinnabarinus as well as abiotic stresses (Lu et al., 2017). This study identified nine SOD genes, which are comparable to the other plants such as C. sativus (9) (Zhou et al., 2017), Solanum lycopersicum (9) (Feng et al., 2016), S. bicolor (8) (Filiz and Tombuloglu, 2015), and M. truncatula (7) (Song et al., 2018). These DcSOD proteins contained the conserved Cu-ZnSOD domain in CSDs. Furthermore, conserved iron/manganese C-terminal domains were also present in DcFSDs and MSDs, as reported in most of the previous studies such as H. annuus L. (Fernández-Ocaña et al., 2011), C. sinensis (Zhou et al., 2019), and C. sativus (Zhou et al., 2017). These results suggest that this gene family contributes to plant development, structure, and responses to abiotic stresses. Salt, drought, heat, and disease tolerance can be improved by altering the expression of certain genes.

The phylogenetic study revealed that D. carota SOD proteins are divided into three distinct subfamilies, namely, Cu/ZnSODs, MnSODs, and FeSODs, which is consistent with Arabidopsis (Kliebenstein et al., 1998), M. truncatula (Song et al., 2018), T. aestivum (Jiang et al., 2019), H. vulgare (Zhang et al., 2021b), and O. sativa L. (Dehury et al., 2013). On the phylogenetic tree, these three subfamilies were split into two classes, namely, Cu/ZnSODs and Fe-MnSODs. MnSODs and FeSODs were grouped, and a high bootstrap value separated them. This indicates that these genes can be classified according to the type of particular domain they contain and that they may have shared ancestral genes. In cotton, it was identified that MSD and FSD families arose from a common ancestor, whereas the CSD subfamily evolved separately. Thus, the two major groups expanded independently (Wang et al., 2017).

The gene structure was significantly comparable between members of the same subfamily in terms of the number of introns and exons, as well as conserved protein motifs, indicating the phylogenetic relationship of D. carota SOD genes was reliable. Moreover, SOD genes from Arabidopsis, D. carota, and O. sativa have a similar distribution in phylogenetic subgroups and intron–exon organizations, indicating that these genes have been highly conserved throughout evolution. Notably, in B. marianensis, a novel SOD has been identified and cloned, which offers significant resistance to high salinity, hydrostatic pressure, chemicals, and adaptability to cold (Li et al., 2020). OsMSD1/3, AtMSD, and DcMSD1/2 had the same motifs. Similarly, AtCSD1, OsCSD3, and DcCS6 had almost the same length and number of exons and introns (Figure 3). These results are comparable to that observed in C. sativus and S. miltiorrhiza, which contain an average of seven exons. Similar results were found in T. aestivum (Jiang et al., 2019), S. bicolor (Filiz and Tombuloglu, 2015), and S. lycopersicum (Feng et al., 2016). Thus, it is speculated that they may share biological functions. Gain/loss of intron/exon, exonization/pseudoexonization, and insertion/deletion cause exon and intron numbers to vary as well as structural variability in distinct genes (Verma et al., 2019).

By comparing DcSOD proteins to their orthologous proteins in other plants (A. thaliana and G. max), the evolution of SOD family was examined. In D. carota, two pairs of SOD genes originated through segmental duplication. Similarly in S. bicolor [54] and watermelon/melon (Kliebenstein et al., 1998), segmental duplication mainly contributed to the expansion of SOD genes. Similarly, SOD genes of G. raimondii and B. napus also showed segmental duplication, which potentially had a significant part in the expansion of respective plant genomes (Wang et al., 2016; Su et al., 2021). In S. lycopersicum, the majority of the genes clustered on a chromosome and experienced tandem duplication. Segmental duplication was also experienced by a few genes. Therefore, in S. lycopersicum both types of duplication resulted in the expansion of this gene family (Kuzniak, 2002). In M. truncatula, neither tandem nor segmental duplication was found; instead, whole-genome duplication was identified, which caused the generation of these genes (Zang et al., 2020). Similarly, for Gossypium, it was proposed that natural selection caused by a number of factors provided the raw material for functional diversification. This resulted in an expansion of this gene family in a relatively short period of time (Lu et al., 2020). This plant experienced the divergence 7–12 Mya, following the duplication of these genes.

In the promoter sequences, cis-elements were predicted to gain a better understanding of the role of DcSOD genes under different environmental conditions (Ijaz et al., 2020). Similar to tomato SlSOD genes, different stress, hormone, and light-responsive elements were identified, which were related to drought, anaerobic induction, and low temperature (Zhang et al., 2015). In cotton, elements involved in heat and cold stress responsiveness, MYB binding site, ABA responsiveness, and drought inducibility were found. It showed the obvious involvement of these genes in abiotic stresses and a potentially significant role in antioxidant activity (Lu et al., 2020). In B. juncea, several stress-responsive elements such as ARE, W-Box, LTR, WUN, Box-W1, and transcription factor binding sites were found, which were possibly activating the mechanisms involved in stress tolerance. In this way, salt stress was tolerated by the activation of different elements (Verma et al., 2019). Different elements that respond to abscisic acid (ABA) and gibberellin were also found (Supplementary Table S4). Therefore, these findings will help further to understand the diverse functions of DcSOD genes under abiotic stresses.

The 3D structures of these D. carota SOD proteins are relatively conserved, which is comparable with conserved domains, phylogeny, and gene structure. These results suggest that D. carota SOD genes may play different roles in different tissues of different genotypes. The fundamental structure of CSD is a β-barrel made up of eight antiparallel strands grouped in Greek key patterns, which helps to stabilize the entire protein structure. On the β-barrels' external side, an active metal site is found. MSD and FSDs' secondary and tertiary structures are also inconsistent with the structure of known proteins, and they work together so that SOD takes up either Mn or Fe for its functions (Verma et al., 2019). H₂O₂ is a ROS that is harmful to plant growth and development. In contrast, SOD is an antioxidant that is involved in the removal of reactive ROS including H₂O₂ (Gill and Tuteja, 2010).

Transcriptome analysis revealed that DcSOD genes had different expression patterns in different tissues and responses to abiotic stresses (dark and light). Similarly, in B. juncea and B. rapa, the expression of SOD genes under heat and drought stresses was found. The genes of both species, B. juncea and B. rapa, were overexpressed under drought stress and very few genes were downexpressed. In heat stress, only two genes were upregulated, while most of the genes showed decreased expression (Verma et al., 2019). The SOD genes, especially MSDs in M. acuminata, showed specific responses to salt, drought, and heat stresses, which depicted their role in the antioxidant activity (Pal et al., 2013). According to expression analysis, five genes (DcFSD2, DcFSD3, DcMSD1, DcMSD2, and DcCSD2) have higher expression in xylem and phloem tissues of D. Carota. DcCSD2, DcMSD1, and DcMSD2 have higher expression, while DcFSD2 and DcFSD3 have minor expression. qRT-PCR analysis revealed that some genes from CSD family were highly activated under cold, heat, salt, and drought stresses. Similarly, in grapevine VvCSD1 was highly expressed in low and high temperatures. Similarly, VvCSD6 had a higher expression in drought stress. Promoter (cis-element) analysis, RNA-seq, and qRT-PCR analysis revealed that most of the genes in the CSD and FSD family, especially DcCSD1, DcCSD2, DcCSD5, and DcFSD2, had a much higher expression for different abiotic stresses. It is hypothesized that different elements and hormones governed the diverse expression level of DcSODs in different stress as well developmental stages. Therefore, these genes can be used for further research as this study has revealed their important role in abiotic stress.

In this study, we found 9 SOD genes in D. carota and further classified them into three subfamilies, namely, MnSODs (DcMSD1–2), FeSODs (DcFSD2–3), and Cu/ZnSODs (DcCSD1–5), and examined their evolutionary relationships, conserved motifs, gene structure, cis-regulatory elements of the promoter regions, and tissue-specific expression pattern. SOD genes have distinct tissue expression levels, indicating that they may have different functions in D. carota growth and development. According to cis-regulatory elements analysis and expression profiles under different stresses, SOD family genes may be associated with response to hormonal and abiotic stress stimuli. Furthermore, we targeted the SOD genes in response to abiotic stresses (cold, heat, salt, and drought) in different tissues and observed that distinct SOD subfamily genes may play a variety of functions in modulating response to abiotic stresses. qRT-PCR results also revealed their higher expression in various stresses. CSD and FSD families showed higher expression levels for abiotic stresses. Collectively, we laid a basis for further functional characterization of D. carota SOD genes in response to abiotic stresses in the future.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

FA conceived the idea. RZ and KF conducted the experiment and collected data. MS and AR helped in analyzing data. ANS, RZ, and KF helped in initial manuscript preparation. FA, KA, HA, RB, and MA provided technically strengthen the basic idea of research during revision process and helped in funding acquition. SF, FA, and ANS proofread and provided intellectual guidance. All authors read and approved the article.

Princess Nourah Bint Abdulrahman University Researchers Supporting Project Number (PNURSP2022R20), Princess Nourah Bint Abdulrahman University, Riyadh, Saudi Arabia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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